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| == Wed PM Practical == SPT (single particle tomography) capabilities are relatively new in EMAN2. They were inspired by Michael Schmid's studies on sub-volume averaging (mostly on viruses), and a stubborn student insisting on doing extensive sub-volume averaging on chaperons. |
== Wednesday - P.M. == |
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| This session will cover the beta version of a small fraction of the SPT | This session will cover the new tools for single particle tomography in EMAN2. Unfortunately, this technique is very memory-intensive and compute-intensive. The 3 gb of ram available on the workshop computers is barely sufficient to do some small examples, and full 3-D alignments of large sets of particles can take many hours of computation. |
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| == OUTLINE == * 1) SPT processing through EMAN2's workflow: e2workflow.py * 2) Sub-volume extraction from tomograms using e2tomoboxer.py * 3) "Preparation" of extracted particles for alignment. [Details later. For a myriad of reasons, it is NOT recommendable to align and average sub-volumes directly after extraction without "preparing" them first]. * 4) Reference based alignment and averaging |
So, for purposes of the tutorial, we will learn how to use the particle picker, and do a couple of small examples which can be completed in the available time. |
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| == SOFTWARE == All necessary software is provided as part of EMAN2. If you don't have EMAN2 installed, you can download the most updated version (for your specific platform, Windows, Linux or Mac), from here: [[http://ncmi.bcm.edu/ncmi/software/counter_222/software_86]] |
== DATA == [[attachment:e2spt_data.zip| e2spt_data.zip|&do=get]] |
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| == RAW DATA == We have prepared sample data for this tutorial. The tomogram in the link below comes from a tilt series of epsilon15 virus particles ''in vitro'', recorded using Zernike phase-plate technology: |
== TUTORIAL DOCUMENT == Not available here for now. Get it through this site: |
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| [[attachment:e15phaseplate.rec]] | http://blake.bcm.edu/emanwiki/Ws2011/Agenda |
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| The following tomogram also comes from a tilt series of epsilon15 viruses ''in vitro'' but was recorded under conventional cryoEM imaging conditions: | == Monstrous command for alignment with e2classaverage3d.py == |
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| [[attachment:e15normal.rec]] | e2classaverage3d.py --input=e15pp_set1_stack.hdf --output=e15pp_set1_aligned.hdf --ref=e15ref_prep_icos_bin2.hdf --npeakstorefine=1 -v 3 --mask=mask.sharp:outer_radius=48 --preprocess=filter.lowpass.gauss:cutoff_freq=.02 --align=rotate_translate_3d:search=10:delta=8:dphi=8:verbose=1:sym=icos --parallel=thread:2 --ralign=refine_3d_grid:delta=3:range=9 --averager=mean.tomo --aligncmp=ccc.tomo --raligncmp=ccc.tomo --shrink=3 --shrinkrefine=3 --savesteps --saveali --iter=8 --normproc=normalize --sym=c1 --keep=0.8 |
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| == BOXING == You have two options for opening the tomogram for purposes of boxing it. |
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| 1) Directly, by typing e2tomoboxer.py followed my the path to the tomogram file on the commandline: | |
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| {{{ e2tomoboxer.py e15phaseplate.rec }}} |
== Monstrous command for alignment with e2tomoallvsall.py == |
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| 2)Or you can launch e2workflow.py from the commandline and access a tomogram through the browser in tomographic menu. | e2tomoallvsall.py -v 1 --path=AVSAs087 --input=CENTEREDvsD8aliVSapo_s087.hdf --shrink=3 --shrinkrefine=2 --iter=87 --mask=mask.sharp:outer_radius=36 --npeakstorefine=16 --preprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --align=rotate_translate_3d:search=4:dphi=12:delta=12 --parallel=thread:24 --ralign=refine_3d_grid:delta=3:range=12:search=2 --averager=mean.tomo --aligncmp=ccc.tomo --raligncmp=ccc.tomo --saveali --savesteps -v 2 --postprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --autocenter --exclusive_class_min=8 --normproc=normalize |
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| [In theory, you can open a tomogram for contemplation purposes by typing: "e2display.py <my_tomogram_path_name_goes_here>" at the commandline, but this is NOT recommendable, unless you have a grossly large (VERY, VERY large) amount of virtual memory on your computer; otherwise, things will crash]. Let's explore the FIRST APPROACH. === OPENING A TOMOGRAM DIRECTLY WITH e2tomoboxer.py === |
== Monstrous command for alignment with e2spt_sim.py == e2tomosim.py --input=../groRef_scaled_bin2.hdf --snr=5 --nptcls=2 --tiltstep=5 --tiltrange=60 --transrange=10 --saveprjs --noiseproc=math.addnoise |
Single Particle Tomography in EMAN2
Wednesday - P.M.
This session will cover the new tools for single particle tomography in EMAN2. Unfortunately, this technique is very memory-intensive and compute-intensive. The 3 gb of ram available on the workshop computers is barely sufficient to do some small examples, and full 3-D alignments of large sets of particles can take many hours of computation.
So, for purposes of the tutorial, we will learn how to use the particle picker, and do a couple of small examples which can be completed in the available time.
DATA
TUTORIAL DOCUMENT
Not available here for now. Get it through this site:
http://blake.bcm.edu/emanwiki/Ws2011/Agenda
Monstrous command for alignment with e2classaverage3d.py
e2classaverage3d.py --input=e15pp_set1_stack.hdf --output=e15pp_set1_aligned.hdf --ref=e15ref_prep_icos_bin2.hdf --npeakstorefine=1 -v 3 --mask=mask.sharp:outer_radius=48 --preprocess=filter.lowpass.gauss:cutoff_freq=.02 --align=rotate_translate_3d:search=10:delta=8:dphi=8:verbose=1:sym=icos --parallel=thread:2 --ralign=refine_3d_grid:delta=3:range=9 --averager=mean.tomo --aligncmp=ccc.tomo --raligncmp=ccc.tomo --shrink=3 --shrinkrefine=3 --savesteps --saveali --iter=8 --normproc=normalize --sym=c1 --keep=0.8
Monstrous command for alignment with e2tomoallvsall.py
e2tomoallvsall.py -v 1 --path=AVSAs087 --input=CENTEREDvsD8aliVSapo_s087.hdf --shrink=3 --shrinkrefine=2 --iter=87 --mask=mask.sharp:outer_radius=36 --npeakstorefine=16 --preprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --align=rotate_translate_3d:search=4:dphi=12:delta=12 --parallel=thread:24 --ralign=refine_3d_grid:delta=3:range=12:search=2 --averager=mean.tomo --aligncmp=ccc.tomo --raligncmp=ccc.tomo --saveali --savesteps -v 2 --postprocess=filter.lowpass.gauss:cutoff_freq=.02:apix=4.401 --autocenter --exclusive_class_min=8 --normproc=normalize
Monstrous command for alignment with e2spt_sim.py
e2tomosim.py --input=../groRef_scaled_bin2.hdf --snr=5 --nptcls=2 --tiltstep=5 --tiltrange=60 --transrange=10 --saveprjs --noiseproc=math.addnoise